Serology assays

ABSTRACT

The invention provides methods and kits for measuring the ability of a test sample to inhibit the binding of a receptor expressed by a pathogen to a host cell ligand of the pathogen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of copending U.S. patentapplication Ser. No. 13/642,347, filed Oct. 19, 2012, which is a 371 ofInternational Application having Serial No. PCT/US2011/032853, filedApr. 18, 2011, which claims the benefit of U.S. Provisional ApplicationNo. 61/342,747, filed Apr. 19, 2010. The entire contents of each ofthese applications are incorporated herein by reference.

FIELD OF THE INVENTION

This application relates to methods and kits for conducting serologyassays. The invention uses arrays of viral receptors to provide a morerobust and scalable alternative to the hemagglutination inhibition (HAI)and microneutralization assays that are typically used to evaluateantibody responses to viral infection or vaccination.

BACKGROUND OF THE INVENTION

The ability to identify influenza-specific antibodies in an individual'sblood is important for a variety of reasons: i) serology testing is usedin epidemiological studies to determine the extent of infection in thepopulation and to characterize the diversity of influenza strains; ii)serological testing of blood from people receiving vaccines is used toassess the efficacy of vaccines; iii) serological testing is also usedto determine if new vaccine strains can be neutralized by the antibodiesgenerated with old vaccine formulations or if the viruses used in thevaccine formulations need to be updated to new strains.

In a serology assay based on conventional ELISA analysis,disease-specific antibodies in a patient sample bind to immobilizedantigens. Antibody binding is detected with a labeled anti-speciesantibody. This “direct binding” format, while simple to carryout, isoften not used for influenza serology because i) conventional directbinding approaches may not differentiate antibody responses to a recentinfection from antibody responses to previous influenza infection; andii) a response in a direct binding assay may not be indicative of theability of an antibody to prevent influenza infection. To circumventthese problems, most influenza serology measurements are carried outusing techniques that focus on identifying antibodies (termed“neutralizing antibodies”) that bind to hemagglutinin or neuraminidaseactive sites and prevent influenza from binding and/or infecting hostcells. Because these active sites change over time as new strainsevolve, neutralizing antibodies tend to be more indicative of recentinfections. In vaccine studies, the generation of neutralizingantibodies is also more indicative of vaccine efficacy.

During the infection process, influenza viruses bind to sialic acidgroups on host cells through a sialic acid receptor, the viralhemagglutinin protein. Hemagglutination inhibition (HAI) assays measurethe ability of antibodies to bind virus hemagglutinin proteins andprevent the binding of virus to red blood cells. The assay end point isvisual. In the absence of antibody, the binding of the virus to redblood cells causes the formation of a red gel-like aggregate(hemagglutination) that fills the test well/tube. In the presence of asufficient neutralizing antibody, hemagglutination is prevented and thered blood cells settle to a small pellet at the bottom of the testwell/tube. Microneutralization assays involve mixing virus with the testantibody sample and combining that mixture with a virus-susceptible cellline. In the absence of antibody, the cells become infected while in thepresence of sufficient neutralizing antibody the cells remainuninfected.

Both the HAI and microneutralization assays require fresh living cellsthat must be collected or grown just in time for the assay. Theseapproaches are cumbersome and can lead to significant day-to-dayvariability in results. Because the end points are binary(hemagglutinated or not; infected or not), a large number of dilutionsmust be run for each sample to identify the concentration of a sampleneeded to cause neutralization. The formats are also inherentlysingleplex. To test the ability of a sample to neutralize multipledifferent virus strains requires multiple independent measurements.

SUMMARY OF THE INVENTION

Therefore, the present invention provides a method for measuring theability of a sample to inhibit the binding of a receptor expressed by apathogen to a host cell ligand of the pathogen, the method comprising(a) contacting a first surface comprising the receptor immobilizedthereto with (i) the sample; and (ii) a particle reagent comprising theligand; and (b) measuring the amount of the particle reagent bound tothe first surface. In one embodiment, the method further includes thestep of incubating the first surface to allow an amount of the particlereagent to bind to the surface. In an alternative embodiment, theinvention provides a method for measuring the ability of a sample toinhibit the binding of a receptor expressed by a pathogen to a host cellligand of the pathogen, the method comprising (a) contacting a firstsurface comprising a receptor immobilized thereto with: (i) the sample;and (ii) a particle reagent comprising ligand, and (b) measuring theamount of the ligand bound to the first surface. In one embodiment, thereceptor is a sialic acid receptor and the ligand is sialic acid.

The contacting step (step (a)) may include (x) incubating the sample andthe first surface; and (y) adding the particle reagent to the mixtureformed in step (x). Alternatively, the first surface is contacted with amixture comprising the sample and the particle reagent. In addition, themethod may also include washing the mixture formed in step (x) prior toadding the particle reagent.

In one embodiment, the sample comprises an antibody and the measuringstep further comprises determining the ability of the antibody toinhibit the binding of the receptor to the ligand. Moreover, the firstsurface may comprise a plurality of different receptors/ligands and themeasuring step comprises measuring the amount of particle reagent boundto each of the different receptors/ligands.

Still further, the method may comprise repeating the method with one ormore control samples having known inhibitory abilities and comparing theconcentration of bound particle reagent measured with the sample to theconcentration of bound particle reagent measured for the one or morecontrol samples to determine the relative inhibitory ability of thesample. For example, the one or more control samples include a negativecontrol sample having no inhibitory component; and/or the one or morecontrol samples include a positive control sample having a definedconcentration of an inhibitory component

In one embodiment, the first surface is positioned within a well of amulti-well assay plate and the sample is measured in the well andoptionally, one or more control sample are measured in one or moreadditional wells of the multi-well assay plate.

The invention also provides a kit for measuring the ability of a sampleto inhibit the binding of a receptor expressed by a pathogen to a hostcell ligand of the pathogen, the kit comprising, in one or morecontainers, vessels or compartments: (a) a first surface comprising thereceptor immobilized thereto; and (b) a particle reagent comprising theligand. Alternatively, the invention provides a kit for measuring theability of a sample to inhibit the binding of a receptor expressed by apathogen to a host cell ligand of the pathogen, the kit comprising, inone or more containers, vessels or compartments: (a) a first surfacecomprising comprising the receptor immobilized thereto; and (b) theparticle reagent comprising the ligand. In one embodiment, the receptoris a sialic acid receptor and the ligand is sialic acid.

The first surface may comprise a patterned array of receptorsimmobilized thereto. The array of receptors may be different strains,subtypes, types, and/or organisms The kit may also include one or morecontrol samples having known inhibitory abilities, and/or a negativecontrol sample having no inhibitory component. One or more controlsamples may include a positive control sample having a definedconcentration of an inhibitory component.

One or more of the components used in the method and/or present in a kitof the invention may be contained within an assay cartridge and/or amulti-well plate. In addition, one or more of the components areprovided in the kit as a reconstitutable dry reagent. In thisembodiment, the dry reagent and the first surface are located in a wellof a multi-well plate and the dry reagent is (i) free standing; (ii)located in a surface of the first surface that does not overlap with thereceptor; or (iii) located on a ledge within the well.

The receptor may be a sialic acid receptor from a virus selected fromthe group consisting of human coronavirus, bovine coronavirus, mousehepatitis virus, equine rhinitis A, Influenza A virus, Influenza Bvirus, and Influenza C virus, Newcastle Disease Virus, murine parvovirusminute virus, reovirus, rotavirus host cell invasion, bluetongue virus,bovine adenovirus serotype 3. In a preferred embodiment, the virus is anInfluenza virus. The measuring step may be used to diagnose if a patientis infected by the pathogen, and/or to determine the efficacy of avaccination protocol for the treatment or prevention of an infectioncharacterized by the presence of the pathogen.

Moreover, the particle reagent may comprise a biological reagentselected from the group consisting of red blood cells, red blood cellvesicles, red blood cell ghosts, membrane fragments, membrane vesicles,proteins, and combinations thereof. In one specific embodiment,biological reagent is mucin. The particle reagent may be cross-linked.In addition, the particle reagent may comprise a coating comprising thebiological reagent, and the particle reagent is selected from the groupconsisting of colloids, beads, and combinations thereof. For example,the particle reagent is comprised of a substance selected from the groupconsisting of polystyrene, polyacrylamide, polypropylene, and latexparticles, silica, alumina, carbon fibrils, and combinations thereof.The particle reagent may be magnetic, conductive and/or semiconductivematerial. In one specific embodiment, the particle reagent comprisescolloidal gold particles. The receptor may be provided in a formselected from the group consisting of whole virus, virus-like particles,purified hemagglutinins, recombinant hemagglutinins, purifiedneuraminidases, recombinant neuraminidases, membrane fragments, membranevesicles, and combinations thereof.

The measuring step may comprise measuring the amount of a detectablelabel attached to the particle reagent, indirectly or directly, e.g.,via a labeled secondary binding reagent. In one embodiment, thesecondary binding reagent is an antibody. The measuring step may includemeasuring a property selected from the group consisting of opticalabsorbance, fluorescence, phosphorescence, chemiluminescence, lightscattering, magnetism, and combinations thereof. In one specificembodiment, the detectable label is an electrochemiluminescence (ECL)label and the measuring step comprises measuring an ECL signal. Themethod optionally comprises correlating the signal with the inhibitoryabilities of the sample. For example, the first surface may be anelectrode and the measuring step further comprises applying a voltagewaveform to the electrode to generate ECL.

DESCRIPTION OF THE FIGURES

FIGS. 1( a)-(c) are schematic representations showing a viral antigenarray (panel (c)) and the use of such an array in a direct serologyassay in which the binding of patient antibodies to antigens on thearray is detected with a labeled anti-species detection antibody (panel(a)) and in a viral receptor neutralization assay in which the presenceof neutralizing patient antibodies against antigen 1 on the arrayprevents binding of,sialic acid receptors on the antigen to labeled redblood cell (RBC) vesicles (panel (b)).

FIGS. 2( a)-(b) show an assay configured according to one embodiment ofthe present invention (panel (a)) and the results obtained from thisassay (panel (b)).

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The articles“a” and “an” are used herein to refer to one or to more than one (i.e.,to at least one) of the grammatical object of the article. By way ofexample, “an element” means one element or more than one element

The invention provides methods and kits for measuring the ability of atest sample to inhibit the binding of a receptor expressed by a pathogento a host cell ligand of the pathogen. In one embodiment, the methodincludes (a) contacting a surface including a receptor immobilizedthereto with the test sample and a particle reagent comprising theligand; and (b) measuring the amount of the particle reagent bound tothe first surface. Alternatively, the surface may have the ligandimmobilized thereto and the method includes contacting the surface witha particle reagent comprising the receptor. Accordingly, the inventionprovides a kit including, in one or more compartments, vessels, orcontainers, (a) a surface including a receptor immobilized thereto; and(b) a particle reagent comprising the ligand; or the kit includes (a) asurface including a ligand immobilized thereto; and (b) a receptor. In apreferred embodiment, the receptor is a sialic acid receptor and theligand is sialic acid.

The invention includes multiplexed formats in which the sample and aparticle reagent comprising a ligand contact a plurality of bindingdomains including immobilized receptors on one or more surfaces, thedifferent binding domains presenting different receptors for that ligand(for example, from different organisms or from different types, subtypesor strains of an organism). The invention also includes multiplexedformats in which the sample and a particle reagent comprising a receptorfrom a pathogen are contacted with a plurality of binding domainsincluding immobilized ligands on one or more surfaces, the differentbinding domains presenting different ligands for that receptor (e.g.,ligands, such as sialic acid, from different host animal species/strainsand/or from different tissue types within a species/strain) Oneembodiment of the invention is illustrated in FIG. 1. FIG. 1( c) depictsan array of receptors from pathogens immobilized to a solid surface (thefigure depicts an array positioned on the bottom of a well of amulti-well plate). The array is contacted with a sample comprisinginhibitors of the binding of viral receptors to their host cell ligands(in this specific example, the inhibitors are depicted as antibodies).The array is also contacted with a particle reagent comprising the hostcell ligand (in this specific example, the particle reagent is a redblood cell). The result of these steps is illustrated in FIG. 1( b). Inthis specific example, the sample contains a neutralizing antibody thatbinds to antigen 1 in the array and inhibits the binding of the particlereagent to the array element containing that antigen. Measurement of theamount of particle reagent bound to a specific array element (directly,through a detectable label on the particle reagent and/or through asecondary reagent that binds the particle reagent) is indicative of theability of the sample to prevent the binding of the receptor in thatarray element to its host cell ligand. The assay format is alsoapplicable to singleplex formats measuring binding to a single viralreceptor. In addition, the same format may be applied to multiplexedassays employing array elements on separate surfaces, such asmultiplexed bead array formats (described in more detail below). Tocontrast the receptor neutralization format to the direct serologyformat, FIG. 1 also illustrates the use of the same antigen array in adirect serology format employing a labeled anti-species antibody as thedetection reagent (FIG. 1( a)).

Because the receptor neutralization assay format measures a functionalactivity that is specific to the receptor, the presence of otherproteins in the preparation does not interfere with the measurement andhigh purity levels are not critical and, e.g., the receptor can beprovided in the form of a crude preparation of the pathogen of interest.In this format, the receptor surface is incubated with particles thatpresent multiple copies of a ligand of the receptor. The presence ofmultiple ligands per particle provides a high avidity interaction to thesurface even if the individual receptor-ligand interactions are lowaffinity. In one embodiment, the surface is contacted with a mixturecomprising the sample and a particle reagent, incubated, and theconcentration of the particle reagent bound to the surface is measured.Alternatively, the surface is first incubated with the sample, theparticle reagent is subsequently added and thereafter ligand-receptorbinding is measured. Optionally, the method may include a washing step,e.g., in which the surface is washed prior to contacting the surfacewith the particle reagent/receptor, and/or prior to the measurementstep. One or more components of the assay may be provided asreconstitutable dry reagents. In specific examples of such anembodiment, a dry reagent may be located within the same container asthe solid surface (e.g., within a well of a multi-well plate) as a freestanding component such as a dry reagent bead, the dry reagent may belocated on a supplemental surface of the solid surface that does notoverlap with a binding domain on said first surface and/or the dryreagent may be located on a ledge within the container. If one or morecomponents are provided in dry form, the addition of sample or otherreagents to the surface can be used to reconstitute the dry reagent foruse in the assay.

In one embodiment, the sample comprises neutralizing antibodies and themeasuring step further comprises determining the ability of theneutralizing antibodies to inhibit the binding of the receptor to theligand. The method may also include repeating the method with one ormore control samples having known inhibitory activities and comparingthe concentration of bound particle reagent measured with the sample tothe concentration of bound particle reagent measured for one or morecontrol samples to determine the relative inhibitory ability of thesample compared to a control. For example, the method may include (a) ananalysis of a negative control sample having no inhibitory component;and/or (b) an analysis of a positive control sample having a definedconcentration of an inhibitory component.

In multiplexed methods of the invention, the assay may include testingthe sample with internal negative and/or positive control receptors. Forexample, an array of receptors from pathogens of interest may alsoinclude a negative control array element that does not bind the ligandor particle reagent. This array element should provide a low assaysignal regardless of the sample. The array may also include a positivecontrol array element that binds the ligand or particle reagent throughan interaction that should not be affected by antibodies against thepathogen of interest. For example the positive array element could be areceptor against the ligand or another component of the particle reagentthat is not derived from the pathogen of interest. Examples of suchreceptors for influenza serology assays include antibodies that bindcomponents of the particle reagent (e.g. antibodies that bind to redblood cells when the particle reagent is derived from red blood cells)or sialic acid receptors that are not derived from influenza (such asplant lectins or hemagglutinins from non-influenza pathogens). Thepositive array element should provide a positive assay signal thatshould not be affected by antibodies against the pathogen of interest.Methods employing the internal control receptors may include measuringthe amount of particle reagent bound to the surfaces/array elementscomprising the control receptors and determining if the amounts arewithin a pre-specified acceptable range.

The assay may be used to diagnose if a patient is infected by a pathogenor to determine the efficacy of a vaccination protocol used for thetreatment or prevention of an infection characterized by the presence ofa pathogen. In one specific example, samples (e.g., sera, plasma, bloodand/or purified antibodies) from patients given a test vaccine aretested according to the methods of the invention for the ability ofantibodies in the samples to prevent a ligand (such as sialic acid) frombinding to multiple different strains of a pathogen (such as differentstrains of influenza). Such an approach may be used to ensure that avaccine provides protection against the diversity of strains to whichvaccinated individuals may be exposed. This approach may also be used totest samples from vaccinated individuals or samples from individualsthat have been exposed in the past to pathogens, to determine if theantibodies generated against these vaccines or older pathogens areuseful in neutralizing one or more newly emerging strains of thepathogen and also to determine whether there is a need to change vaccineformulations to address genetic changes in the pathogen. Preferably,these approaches are carried out in a multiplexed format using themultiplexed methods and kits of the invention.

In another example, samples from individuals (which may have beenselected based on known exposure to a pathogen) are tested for theirability to inhibit the receptor from multiple strains of the pathogen toprovide an epidemiological characterization of the circulating strainsof the pathogen in a population.

In another example, a particle reagent comprising a receptor from apathogen is contacted with one or more surfaces comprising differentligands (preferably, in a multiplexed format of the invention) and thebinding of the particle reagent to the ligands is measured to determinethe ligand specificity of the pathogen receptor. For example, differentsialic acid structures (which may be synthetically created or isolatedfrom different animal species/strains or different tissue types from ananimal species/strain) are tested to determine the sialic acidspecificity of an influenza virus hemagglutinin.

In a preferred embodiment, the receptors are sialic acid receptors thatthe corresponding ligand is sialic acid. Sialic acid is a generic termfor the N- or O-substituted derivatives of neuraminic acid, anine-carbon monosaccharide. Sialic acids are found widely distributed inanimal tissues and in bacteria, especially in glycoproteins andgangliosides. The amino group bears either an acetyl or a glycolylgroup. Sialic acid structure may differ from one strain, type, subtypeand/or organism to another.

Sialic acid receptors may be the same or different and the assay mayinvolve measuring a binding interaction between sialic acid moietiesdifferent sialic acid receptors. In certain embodiments, the receptor isa sialic acid receptor from a virus selected from the group consistingof human coronavirus, bovine coronavirus, mouse hepatitis virus, equinerhinitis A, Influenza A virus, Influenza B virus, and Influenza C virus,Newcastle Disease Virus, murine parvovirus minute virus, reovirus,rotavirus host cell invasion, bluetongue virus, bovine adenovirusserotype 3. In a specific embodiment, the virus is an Influenza virus.The receptor may be presented as a whole virus, virus-like particles,purified hemagglutinins, recombinant hemagglutinins, purifiedneuraminidases, recombinant neuraminidases, membrane fragments, membranevesicles, and combinations thereof.

For hemagglutinin assays, one convenient source of sialic coatedparticles is red blood cells. Optionally, to provide stable reagentsthat can be prepared in large quantities and stored for later use, itmay be preferable to use red blood cell ghosts, membrane fragments orvesicles which may, optionally, be cross-linked. Alternatively, cells,membrane fragments or vesicles from other cell types may be used. In oneembodiment cells from or derived from the epithelia of the respiratoryor gastro-intestinal tracts are used because these cells also tend todisplay sialic acids. The predominant sialic acid structure can bevaried by selecting cells from different locations in the respiratory orgastro-intestinal tracts. One skilled in the art will be able to selectcells that bind the viruses of interest and/or screen through cell typeto identify cells with appropriate binding characteristics. Moreover, itis known that red blood cells from different species may have differentbinding affinities for viral particles. Therefore, it may be beneficialto screen through cells from different species to find cells withoptimal binding characteristics. In another approach, proteinspresenting multiple sialic acids (e.g., fetuins, mucins and α-acidglycoproteins) are used instead of particles or are coated on particlesto provide the particle reagent. In yet another approach, syntheticsialic acids or conjugates of synthetic sialic acids and othercomponents (such as lipids or synthetic or biological polymers includingproteins, polysacharrides) are used and are coated or chemically coupledto particles to prepare the particle reagents.

The particle reagent comprising a host cell ligand may include abiological reagent selected from the group consisting of red bloodcells, red blood cell vesicles, red blood cell ghosts, membranefragments, membrane vesicles, proteins, and combinations thereof. In oneembodiment, the particle reagent is cross-linked. The particle reagentitself may be a biological material, e.g., red blood cells, red bloodcell vesicles, red blood cell ghosts, membrane fragments, membranevesicles, proteins, and combinations thereof, or it may be comprised ofanother, e.g., synthetic material, that may be coated with a biologicalreagent. The particle itself may consist of colloids, beads, andcombinations thereof. In one embodiment, the particle reagent iscomprised of a substance selected from the group consisting ofpolystyrene, polyacrylamide, polypropylene, and latex particles, silica,alumina, carbon fibrils, and combinations thereof. The particle may bemagnetic, conductive and/or semiconductive. In one specific embodiment,the particle reagent consists of colloidal gold particles. Where themethod employs a ligand immobilized on a surface, the immobilizedcomponent may be formed by immobilizing a particle reagent comprisingthe ligand as described above.

In one embodiment the particle is between 10 nm and 1 mm in diameter. Inanother embodiment, the particle is between 10 nm and 0.02 mm indiameter. In another embodiment, the particle is between 40 nm and 1000nm in diameter.

In one embodiment, the particle is a labeled cell, cell ghost or a smallunilamellar vesicle (SUV) prepared from a cell, for example, from a redblood cell. Such particles may be provided as stable or frozenpreparations. For a discussion of the preparation of stable frozen orlyophilized vesicle preparations, see, e.g., Mohammed A R et al.,Methods, 2006, 40, 30-38. For a discussion of approaches to stabilizingred blood cells for storage, see, e.g., Torok, Z. et al., CellPreservation Technology, 2005, 3: 96-111.

In one embodiment, the particle may be coated with a biological membranewhich is displayed on the particle. Such membrane coated particles maybe prepared by incubating particles with membranes (e.g., cells,membrane fragment and membrane vesicles). The approach may be used withmetal particles, high surface area particles, e.g., silica or otherparticles with oxide-based surfaces, and particles with a hydrophobicsurface. In a preferred embodiment, the particle is a silica particle.During this coating process, the membrane orientation is reversed.Incubating red blood cell ghosts with silica particles, therefore, leadsto silica particles presenting the cytoplasmic side of the red bloodcell membranes (Kaufmann S et al., ChemPhysChem, 2003, 4, 699-704).

The invention includes a method for coating particles with membranes soas to display the outside surface of a cell by incubating particles(e.g., silica particles) with inside-out membranes, i.e., any vesicleformed from disrupted cell or other membranes in which the face of themembrane that was originally on the outside surface is now on theinside. The invention also includes such particles and their use in theassays of the invention. Inside out vesicles for use in the method canbe formed using established methods such as those described in Palmgrenet al., Plant Physiol, 1990, 92, 871-880 or Walsh et al., Biochemistry,1976, 15, 3557-63. Specific methods for preparing inside out red bloodcell ghosts for use in forming coated particles presenting the outersurface of red blood cell particles are described, e.g., in Steck T L etal., Science, 1970, 168, 255-257).

In an alternative embodiment, vesicles or undefined or random membraneorientation are used to coat particles, such that the particles presentboth internal and external membrane proteins. Such vesicles include, butare not limited to, vesicles prepared by sonication or by removingdetergent from detergent from detergent-solubilized membranes. In oneexample, red blood cell SUVs presenting both internal and externalmembrane proteins (e.g., SUVs prepared by sonication) are used as themembrane protein source to create particles presenting a randomdistribution of internal and external red blood cell membrane proteins.

In another alternate embodiment, cell or membrane Lysates solubilized indetergent are covalently coupled to particles. In one example, thecoupling is by using standard EDC coupling protocols to attachcomponents of the lysates to carboxyl modified latex particles, althoughother know functionalized particles and coupling chemistries may beused. Where maintaining protein structure is critical, a non-denaturingdetergent is, preferably, used. For secondary structure independentbinding groups such as sugars, denaturing or non-denaturing detergentsmay be used. Useful detergents include non-ionic detergents such asTriton X-100. In one specific example, red blood cell surface proteinsare coupled to a particle.

In another alternate embodiment, labeled soluble proteins are used inthe assay methods of the invention instead of the labeled particles. Inone example, soluble sialic acid containing proteins (e.g., fetuins,mucins or a-acid glycoproteins) are labeled and used in monomeric form.The soluble proteins may also be aggregated by chemical cross-linking(e.g., using established methods such gluteraldehyde cross-linking) toform larger aggregates with higher avidity. Alternatively, the solubleproteins (e.g., the sialic acid containing proteins as described above)may be adsorbed or covalently linked to particles. By way of example,the coating of particles with mucin and fetuin by adsorption tounmodified latex particles or by covalent coupling to carboxy-modifiedlatex particles is described in Szoke et al., J. Med. Microbiol., 1996,45, 338-343, as is the use of these particles in latex-agglutinationassays.

In one preferred embodiment, the invention includes immobilization of amaterial, e.g., a receptor or particle reagent, onto one or more assayelectrodes. Such electrodes may be incorporated into a variety ofdifferent assay modules suitable for carrying out assays, e.g., assayplates, cassettes, cartridges, devices, etc. Preferably, the electrodeis incorporated in the wells of a multi-well assay plate. The assayregion or module (e.g., a given well of a multi-well plate) may alsocomprise additional electrodes. Preferably at least one electrode in anassay region or module (or a well of a multi-well plate) is suitable foruse as a working electrode in an electrode induced luminescence assay,at least one electrode is suitable for use as counter electrode in anelectrode induced luminescence assay. Optionally, there is at least oneelectrode that is suitable for use as a reference electrode (e.g., in athree electrode electrochemical system). Preferably, no referenceelectrode is included.

The receptors or particles can be immobilized on a surface via a varietyof interactions including non-specific adsorption (e.g., vianon-specific ionic, hydrogen bonding, polar, Van der Waals and/orhydrophobic interactions), covalent bonding, and/or specific bindinginteractions between binding partners (e.g., ligand/receptor,antibody/hapten, nucleic acid hybridization, biotin/avidin,biotin/streptavidin, lectin/sugar, metal/ligand, etc.). Preferably, thematerial is immobilized directly onto the surface, more preferablywithout the use of an immobilization agent (e.g., a dye, trehalose,etc.). Alternatively, a receptor or particle reagent is immobilized viathe binding of a receptor or particle reagent components to antibodiesimmobilized on the surface. In another preferred embodiment, a receptoror particle reagent is immobilized via the binding of a labeledcomponent of the receptor or particle reagent (e.g., a biotin or haptenlabeled moiety, protein or sugar) to a binding reagent (e.g.,streptavidin, avidin or an antibody) immobilized on a surface. Thesurface may include a spacer layer between the surface and animmobilized receptor or particle reagent. Such a layer may function,e.g., as a chemical linker for holding the layer to the surface and/oras a hydrophilic spacer volume.

In some embodiments of the invention, the immobilized receptor orparticle reagent are cross-linked so as to provide greater stability.Cross-links may include i) cross-links between receptor/particlecomponents (e.g., lipids, proteins and/or sugars) and chemical moietieson the electrode surface and ii) cross links between receptor/particlecomponents themselves. Cross-linking may be accomplished by a variety oftechniques, e.g., techniques known in the arts of tissue fixing, samplepreparation for microscopy, bioconjugate chemistry, affinity-labelingand the preparation of cross-linked lipid membranes. Usefulcross-linking reagents include cross-linking reagents that comprise oneor more functional groups capable of reacting with components of areceptor/particle layer or an electrode surface (e.g., imidoesters,active esters such as NHS esters, maleimides, α-halocarbonyls,disulfides such as pyridyldithiols, carbodiimides, arylazides, amines,thiols, carboxylates, hydrazides, aldehydes or ketones, activecarbamates, glyoxals, etc.). In some applications it may be advantageousto use photo-reactive cross-linkers (such as arylazides) so as to bettercontrol the cross-linking process. Exemplary cross-linking agentsinclude homo- and hetero-bifunctional cross-linking agents such as thosesold by Pierce Chemical Co. and/or described in the 1994 Pierce Catalogand Handbook (Pierce Chemical Co., Rockford, Ill., 1994), the chaptersrelating to cross-linking agents hereby incorporated by reference. Lipidmonolayers and bilayers may be cross-linked by chemically cross-linkinglipid head-groups and/or tail groups (e.g., by including lipids withtails comprising photochemically cross-linkable groups such as alkene oralkyne groups and/or by including lipids that can span bilayer lipidmembranes). See, U.S. Pat. No. 5,637,201, hereby incorporated byreference.

According to one embodiment, the assay surface is incorporated in anassay module, e.g., an electrode located in one or more wells of amulti-well plate. Suitable assay modules, including multi-well assaymodules, and method of using and systems incorporating the same are setforth in U.S. application Ser. No. 10/185,274, entitled “Assay Plates,Reader Systems and Methods for Luminescence Test Measurements”, filedJun. 28, 2002 (see Sections 3, 4 and 5.1-5.6), hereby incorporated byreference. According to one preferred embodiment of the invention, anassay module or plate comprises one or more (preferably two or more, 6or more, 24 or more, 96 or more, 384 or more, 1536 or more or 9600 ormore) assay wells, assay chambers and/or assay domains (e.g., discretelocations on a module surface where an assay reaction occurs and/orwhere an assay signal is emitted; typically an electrode surface,preferably a working electrode surface). According to a particularlypreferred embodiment, the assay plate is a multi-well assay plate havinga standard well configuration (e.g., 6 well, 24 well, 96 well, 384 well,1536 well, 6144 well or 9600 well). Alternatively, the assay surface isincorporated in an assay cartridge, e.g., as described in U.S.application Ser. No. 10/744,726, entitled “Assay Cartridges and Methodsof Using the Same, filed Dec. 23, 2003, and U.S. Application Ser. No.61/284,276, entitled “Assay Cartridges and Methods of Using the Same,filed Dec. 16, 2009, the disclosures of which are incorporated herein byreference.

The measurement of the binding interaction between a receptor/ligand mayconsist of measuring the amount of a detectable label attached, directlyor indirectly, to the surface via a binding interaction between a ligandand a particle reagent. The measurement may also reflect thepresence/absence of neutralizing antibodies in a test sample whichinterfere with binding between the surface immobilized particle/receptorand the free receptor/particle in solution. Receptor-ligand binding maybe measured using any of a number of techniques available to the personof ordinary skill in the art, e.g., direct physical measurements (e.g.,mass spectrometry) or binding assays (e.g., immunoassays, agglutinationassays and immunochromatographic assays). The method may also comprisemeasuring a signal that results from a chemical reactions, e.g., achange in optical absorbance, a change in fluorescence, the generationof chemiluminescence or electrochemiluminescence, a change inreflectivity, refractive index or light scattering, the accumulation orrelease of detectable labels from the surface, the oxidation orreduction or redox species, an electrical current or potential, changesin magnetic fields, etc. Suitable detection techniques may detectbinding events by measuring the participation of labeled bindingreagents through the measurement of the labels via theirphotoluminescence (e.g., via measurement of fluorescence, time-resolvedfluorescence, evanescent wave fluorescence, up-converting phosphors,multi-photon fluorescence, etc.), chemiluminescence,electrochemiluminescence, light scattering, optical absorbance,radioactivity, magnetic fields, enzymatic activity (e.g., by measuringenzyme activity through enzymatic reactions that cause changes inoptical absorbance or fluorescence or cause the emission ofchemiluminescence). Alternatively, detection techniques may be used thatdo not require the use of labels, e.g., techniques based on measuringmass (e.g., surface acoustic wave measurements), refractive index (e.g.,surface plasmon resonance measurements), or the inherent luminescence ofan analyte or direct visualization of particles.

Binding assays for measuring receptor/ligand interactions may use solidphase or homogenous formats. Suitable assay methods include sandwich orcompetitive binding assays. Examples of sandwich immunoassays aredescribed in U.S. Pat. No. 4,168,146 and U.S. Pat. No. 4,366,241, bothof which are incorporated herein by reference in their entireties.Examples of competitive immunoassays include those disclosed in U.S.Pat. No. 4,235,601, U.S. Pat. No. 4,442,204 and U.S. Pat. No. 5,208,535,each of which are incorporated herein by reference in their entireties.

Multiple receptor/ligand interactions may be measured using amultiplexed assay format, e.g., multiplexing through the use of bindingreagent arrays, multiplexing using spectral discrimination of labels,multiplexing of flow cytometric analysis of binding assays carried outon particles, e.g., using the Luminex® xMAP technology. Suitablemultiplexing methods include array based binding assays using patternedarrays of immobilized receptors/particles directed associated with thepathogen. Various approaches for conducting multiplexed assays have beendescribed (See e.g., US 20040022677; US 20050052646; US 20030207290; US20030113713; US 20050142033; and US20040189311, each of which isincorporated herein by reference in their entireties. One approach tomultiplexing binding assays involves the use of patterned arrays ofbinding reagents, e.g., U.S. Pat. Nos. 5,807,522 and 6,110,426;Delehanty J-B., Printing functional protein microarrays usingpiezoelectric capillaries, Methods Mol. Bio. (2004) 278: 135-44; Lue R Yet al., Site-specific immobilization of biotinylated proteins forprotein microarray analysis, Methods Mol. Biol. (2004) 278: 85-100;Lovett, Toxicogenomics: Toxicologists Brace for Genomics Revolution,Science (2000) 289:536-537; Berns A, Cancer: Gene expression indiagnosis, nature (2000), 403,491-92; Walt, Molecular Biology:Bead-based Fiber-Optic Arrays, Science (2000) 287: 451-52 for moredetails). Another approach involves the use of binding reagents coatedon beads that can be individually identified and interrogated. See e.g.,WO 9926067, which describes the use of magnetic particles that vary insize to assay multiple analytes; particles belonging to differentdistinct size ranges are used to assay different analytes. The particlesare designed to be distinguished and individually interrogated by flowcytometry. Vignali has described a multiplex binding assay in which 64different bead sets of microparticles are employed, each having auniform and distinct proportion of two dyes (Vignali, D., “MultiplexedParticle-Based Flow Cytometric Assays” J. ImmunoL Meth. (2000) 243:243-55). A similar approach involving a set of 15 different beads ofdiffering size and fluorescence has been disclosed as useful forsimultaneous typing of multiple pneumococcal serotypes (Park, M. K etal., “A Latex Bead-Based Flow Cytometric Immunoassay Capable ofSimultaneous Typing of Multiple Pneumococcal Serotypes (MultibeadAssay)” Clin. Diag. Lab ImmunoL (2000) 7: 4869). Bishop, J E et al. havedescribed a multiplex sandwich assay for simultaneous quantification ofsix human cytokines (Bishop, L E. et al., “Simultaneous Quantificationof Six Human Cytokines in a Single Sample Using Microparticle-based FlowCytometric Technology,” Clin. Chem (1999) 45:1693-1694).

The method of the present invention may be conducted in a single assaychamber; such as a single well of an assay plate or an assay chamberthat is an assay chamber of a cartridge. The assay modules, e.g., assayplates or cartridges or multi-well assay plates), methods andapparatuses for conducting assay measurements suitable for the presentinvention are described for example, in US 20040022677; US 20050052646;US 20050142033; US 20040189311, each of which is incorporated herein byreference in their entireties. Assay plates and plate readers are nowcommercially available (MULTI-SPOT® and MULTI-ARRAY® plates and SECTOR®instruments, Meso Scale Discovery, a division of Meso Scale Diagnostics,LLC, Gaithersburg, Md.).

According to one embodiment, the kit of the present invention includesone or more of the assay components in one or more locations on asurface and/or in a device used in an assay method, preferably in dryform. In a preferred embodiment, one or more of the components of theassay are provided in the kit as a reconstitutable dry reagent, whereinthe dry reagent is free standing; or located, on a supplemental surfaceof the first surface that does not overlap with a binding domain on thefirst surface. The assay components may be provided in separatecontainers, vessels or compartments. In one embodiment, the kit includesa container comprising one or more reagents, buffers, co-reactants,blocking agents and/or stabilizing agents.

One preferred embodiment relates to a kit for use in conductingelectrode induced luminescence assays (preferablyelectrochemiluminescence assays) comprising an assay plate, preferably amulti-well assay plate, or an assay cartridge, including a first surfacehaving a particle/reagent immobilized thereto, a corresponding reagentselected from a particle reagent or a receptor, and at least one assaycomponent selected from the group consisting of at least one luminescentlabel (preferably electrochemiluminescent label) and at least oneelectrochemiluminescence coreactant). The kit may further includeadditional buffers and/or blocking reagents for use in the method of thepresent invention.

According to another embodiment, the kit is adapted for multiple assays.Preferably, the kit further comprises an additional assay reagent foruse in an additional assay, the additional assay selected from the groupconsisting of radioactive assays, enzyme assays, chemical colorimetricassays, fluorescence assays, chemiluminescence assays and combinationsthereof.

EXAMPLES

The following example is illustrative of some of the kits and methodsfalling within the scope of the present invention. It is, of course, notto be considered in any way limitative of the invention. Numerouschanges and modification can be made with respect to the invention byone of ordinary skill in the art without undue experimentation.

Example 1 Viral Receptor Neutralization Assay

In this set of experiments, the ability of an H1 specific goatpolyclonal antibody to inhibit the binding of an H1N1 virus to guineapig red blood cell vesicles was tested. The red blood cell vesicles weremade by preparing guinea pig red blood cell ghosts and sonicating theghosts to form vesicles. H1N1 virus particles were labeled by labeling awhole virus preparation with an ECL label (SULFO-TAG™ NHS ester, MesoScale Diagnostics, LLC, Gaithersburg, Md. 20877). When the vesicles wereimmobilized by passive adsorption on carbon ink electrodes, theyretained the ability to bind the red blood cell vesicles via theinteraction of the viral hemagglutinin with the red blood cell sialicacids. The extent of binding was measured usingelectrochemiluminescence. FIG. 2( a) shows the configuration of theassay and FIG. 2( b) shows the inhibition of the binding reaction thatoccurs when increasing amounts of a polyclonal anti-H1 antibody wereintroduced. In this experiment, red blood cell vesicles were immobilizedand the whole virus was labeled, but the reverse orientation, i.e.,immobilized virus and labeled red blood cell vesicles, will work aswell.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of themethod in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theclaims. Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

What is claimed is:
 1. A method for measuring the ability of a sample toinhibit the binding of a receptor expressed by a pathogen to a host cellligand of said pathogen, said method comprising (a) contacting a firstsurface comprising said receptor immobilized thereto with (i) saidsample; and (ii) a particle reagent comprising said ligand; and (b)measuring the amount of said particle reagent bound to said firstsurface.
 2. The method of claim 1 wherein said receptor is a sialic acidreceptor and said ligand is sialic acid.
 3. The method of claim 1wherein said contacting step (a) comprises the following steps in order:(x) incubating said sample and said first surface; and (y) adding saidparticle reagent to the mixture formed in step (x).
 4. The method ofclaim 3, further comprising washing the mixture formed in step (x) priorto adding said particle reagent.
 5. The method of claim 1, wherein saidfirst surface is contacted with a mixture comprising said sample andsaid particle reagent.
 6. The method of claim 1 wherein said samplecomprises an antibody and said measuring step further comprisesdetermining the ability of said antibody to inhibit the binding of saidreceptor to said ligand.
 7. The method of claim 1 wherein said firstsurface comprises a plurality of different receptors and said measuringstep comprises measuring the amount of particle reagent bound to each ofsaid different receptors.
 8. The method of claim 1 wherein said methodfurther comprises repeating said method with one or more control sampleshaving known inhibitory abilities and comparing the concentration ofbound particle reagent measured with said sample to the concentration ofbound particle reagent measured for said one or more control samples todetermine the relative inhibitory ability of said sample.
 9. The methodof claim 8 wherein said one or more control samples include a negativecontrol sample having no inhibitory component.
 10. The method of claim 8wherein said one or more control samples include a positive controlsample having a defined concentration of an inhibitory component. 11.The method of claim 8 wherein said first surface is positioned within awell of a multi-well assay plate and said sample is measured in saidwell and said one or more control sample are measured in one or moreadditional wells of said multi-well assay plate.
 12. The method of claim1 wherein said receptor is a sialic acid receptor from a virus selectedfrom the group consisting of human coronavirus, bovine coronavirus,mouse hepatitis virus, equine rhinitis A, Influenza A virus, Influenza Bvirus, and Influenza C virus, Newcastle Disease Virus, murine parvovirusminute virus, reovirus, rotavirus host cell invasion, bluetongue virus,bovine adenovirus serotype
 3. 13. The method of claim 12 wherein saidvirus is an Influenza virus.
 14. The method of claim 1 wherein saidmeasuring step is used to diagnose if a patient is infected by saidpathogen.
 15. The method of claim 1 wherein said measuring step is usedto determine the efficacy of a vaccination protocol for the treatment orprevention of an infection characterized by the presence of saidpathogen.
 16. The method of claim 1 wherein said particle reagentcomprises a biological reagent selected from the group consisting of redblood cells, red blood cell vesicles, red blood cell ghosts, membranefragments, membrane vesicles, proteins, and combinations thereof. 17.The method of claim 1 wherein said particle reagent is cross-linked. 18.The method of claim 16 wherein said particle reagent comprises a coatingcomprising said biological reagent.
 19. The method of claim 18 whereinsaid particle reagent is selected from the group consisting of colloids,beads, and combinations thereof.
 20. The method of claim 19 wherein saidparticle reagent is comprised of a substance selected from the groupconsisting of polystyrene, polyacrylamide, polypropylene, and latexparticles, silica, alumina, carbon fibrils, and combinations thereof.21. The method of claim 19 wherein said particle reagent is magnetic.22. The method of claim 19 wherein said particle reagent is comprised ofa conductive and/or semiconductive material.
 23. The method of claim 22wherein said particle reagent comprises colloidal gold particles. 24.The method of claim 1 wherein said receptor is provided in a formselected from the group consisting of whole virus, virus-like particles,purified hemagglutinins, recombinant hemagglutinins, purifiedneuraminidases, recombinant neuraminidases, membrane fragments, membranevesicles, and combinations thereof.
 25. The method of claim 1 whereinsaid measuring step comprises measuring the amount of a detectable labelattached to said particle reagent.
 26. The method of claim 25 whereinsaid detectable label is attached to said particle reagent directly. 27.The method of claim 25 wherein said detectable label is attached to saidparticle reagent via a labeled secondary binding reagent.
 28. The methodof claim 27 wherein said secondary binding reagent is an antibody. 29.The method of claim 1 wherein said measuring step comprises measuring aproperty selected from the group consisting of optical absorbance,fluorescence, phosphorescence, chemiluminescence, light scattering,magnetism, and combinations thereof.
 30. The method of claim 25 whereinsaid detectable label is an electrochemiluminescence (ECL) label andsaid measuring step comprises measuring an ECL signal.
 31. The method ofclaim 30 wherein said method further comprises correlating said signalwith the inhibitory abilities of said sample.
 32. The method of claim 30wherein said first surface is an electrode and said measuring stepfurther comprises applying a voltage waveform to said electrode togenerate ECL.
 33. The method of claim 16 wherein said biological reagentis mucin.